Screening Of Differentially Expressed Proteins In Pulmonary Tuberculosis Associated Neutrophils And Exploring Its Application In The Diagnosis Of Pulmonary Tuberculosis

Objective:With Healthy(Healthy Control,HC)of peripheral blood neutrophils proteome,screening and identification of Active TB patients(Active tuberculosis,ATB)of peripheral blood neutrophils(NEU,Neutrophil)differentially expressed proteins,selection and Neutrophil degranulation or activation related differentially expressed proteins,discusses its applied value in the diagnosis of tuberculosis(TB),offers a new protein markers for the diagnosis of tuberculosis.Method:Inclusion and differential proteomics detection of subjects: 15 ATB patients and15 healthy subjects were selected and included,and peripheral blood was collected and purified Neu cells from peripheral blood by immunomagnetic bead negative selection method.Will be incorporated into the ATB patients and healthy physical examination is divided into three groups,each each group included 5 patients with ATB or physical examination of NEU cells mix sample,and the Label-free proteomics technology identification and assessment of each sample group of proteins,according to differentially expressed multiples(a Fold change,FC)1.5 or higher for the standard screening of ATB differentially expressed proteins in patients with peripheral blood NEU.2.The bioinformatics analysis of differentially expressed proteins and the determination of candidate target protein: bioinformatics analysis was carried out on the differences in protein,according to the difference in expression multiples,whether it is secreted protein,is associated with NEU cell degranulation and activation function,whether there are goods kits,choose and NEU degranulation or activation type secrete proteins that function as a further analysis of the candidate target protein.3.Candidate serum level of target protein detection: collecting ATB patients and serum samples of HC,scattering turbidimetry with ELISA or rate of candidate target protein levels were detected and compared,clear each candidate target protein in patients with tuberculosis(TB)in the serum level change,choice and HC level exists significant difference of serum protein as diagnosis effectiveness analysis of target protein.4.Efficiency analysis of target protein in ATB diagnosis:Select and ATB patients,Latent tuberculosis infection(Latent tuberculosis infection,LTBI),lung cancer,lung cancer patients,LC)and HC,by ELISA and rate scattering turbidimetric method to detect the research object of each target protein in the serum levels,comparing the target protein level differences in different groups the research object,combined with the subjects work curve(ROC)analysis and about an index to evaluate the target proteins and their combination in the diagnosis of active TB.Results:1.Results of differential proteomics: A total of 2313 proteins were isolated and identified from peripheral blood NEU from ATB patients and healthy controls using Label-free proteomics technique,of which 1326 proteins were quantifiable.Based on the criteria of Fold change(Fc)≥1.5,the protein expression profile data of NEU samples were analyzed.A total of 456 proteins with Fold change ≥1.5 were selected,among which 374 proteins were up-regulated and 82 proteins were down-regulated.2.The choice of candidate target protein results: through analyzing the differentially expressed proteins in bioinformatics,according to whether these differences in protein is secreted protein,is associated with NEU degranulation and activation process,this study selected the NEU degranulation or activation related differentially expressed protein secretion of 54,which raised expressions of 34,lower expression of 20.Based on the availability of commercial detection kits,six proteins significantly down-regulated in NEU of ATB patients were selected as candidate target proteins for further analysis in this study,including AHSG,AACT,AGP2,A1 BG,C3,AAT,AAG and HP.3.The results of the detection of the above candidate target proteins showed that compared with the healthy control group,the expression level of AHSG in serum of tuberculosis patients was significantly decreased,while the levels of AACT,AGP2,A1 BG,C3,AAT,AAG and HP were significantly increased,and the differences were of significant scientific significance.4.The results of training concentration verification were as follows:(1)The analysis results with HC as the control group showed that the levels of serum AHSG were significantly down-regulated,while the levels of AACT,C3,AAT,AAG and HP were significantly increased in the patients with tuberculosis,and the differences were statistically significant.(2)All subjects in the non-tuberculosis group were taken as the control group.The results showed that the expressions of AACT,C3,AAT,AAG and HP were up-regulated in the tuberculosis group,while the expressions of AHSG were down-regulated in the tuberculosis group,with statistically significant differences.(3)The serum levels of AHSG,C3,AAT and AAG in tuberculosis patients were significantly different from those in HC,LC and LTB groups,while the serum levels of AACT and HP in tuberculosis patients were not significantly different from those in LC group.5.ROC curve analysis showed that the AUC values of AHSG,AACT,C3,AAT,AAG and HP in the diagnosis of tuberculosis were 0.845、0.769、0.736、0.825、0.708 and 0.674,respectively,when all the subjects in the non-tuberculosis group were taken as the control group.When AHSG,AAT and AAG were combined pairwise,the combined efficiency of AHSG and AAT was the best,the AUC was 0.912,and the sensitivity and specificity were 82.81% and 89.42%,respectively.The efficiency of AHSG,AAT and AAG combined diagnosis of ATB was not significantly higher than that of AHSG and AAT combined diagnosis.Conclusion:This study applies the Label-free quantitative proteomic technology to establish the ATB patients with HC NEU controls peripheral blood cells of protein expression differences between the spectrum,and through the sieve out some bioinformatics analysis related to NEU cells activated degranulation,and other functions of differentially expressed proteins,validated by candidate protein expression and analyze its diagnostic performance,found that both candidate protein AHSG and AAT has high diagnostic performance,can effectively distinguish between ATB patients,patients with LC,LTBI and HC contrast,is expected to become the new potential biomarkers TB diagnosis.